Extending Top-Down Mass Spectrometry to Proteins with Masses Greater Than 200 Kilodaltons

Abstract

For characterization of sequence and posttranslational modifications, molecular and fragment ion mass data from ionizing and dissociating a protein in the mass spectrometer are far more specific than are masses of peptides from the protein's digestion. We extend the approximately 500-residue, approximately 50-kilodalton (kD) dissociation limitation of this top-down methodology by using electrospray additives, heated vaporization, and separate noncovalent and covalent bond dissociation. This process can cleave 287 interresidue bonds in the termini of a 1314-residue (144-kD) protein, specify previously unidentified disulfide bonds between 8 of 27 cysteines in a 1714-residue (200-kD) protein, and correct sequence predictions in two proteins, one with 2153 residues (229 kD).

Nonergodicity in electron capture dissociation investigated using hydrated ion nanocalorimetry

“…For Ca(H 2 O) 4 ϩ , both levels of theory indicate that Ca ϩ is solvated by four water molecules in plane with the metal ion, although we found a D 2h symmetry to be slightly lower in energy than the C s -symmetry structure with distorted O-Ca-O bond angles identified previously. For Ca(H 2 O) 5 ϩ and Ca(H 2 O) 6 ϩ , our calculations indicate that all water molecules in the lowest-energy structure coordinate directly to the metal ion, whereas the previous calculations indicate that structures with four inner-shell water molecules are lowest in energy. Because Watanabe et al [67] reported relative free energies for structures, it is difficult to directly compare these results.…”

“…Clemmer and coworkers elegantly demonstrated that combining on-line liquid chromatography (LC) with ion mobility spectrometry and MS can greatly improve separations without increasing analysis times over LC/MS alone [2][3][4]. In contrast, the "top-down" approach to protein characterization has the advantage that de novo sequencing, including the identification and structural localization of labile posttranslational modifications, can be done directly on protein mixtures without proteolysis [5,6]. This top-down approach has greatly benefited from the development of electron capture dissociation (ECD), a method pioneered by McLafferty and coworkers [6][7][8][9].…”

“…A measure of the internal energy can also be obtained from the abundances of fragment ions formed via consecutive reaction pathways with known critical formation energies [42][43][44][45]. For example, activation of Fe(CO) 5 ϩ· can result in sequential loss of CO molecules with critical energies ranging from 1.15 eV for the loss of the first CO molecule to 7.58 eV for the loss of all five CO molecules; formation of FeC ϩ requires 15.7 eV [43]. The abundances of the fragment ions formed by activation of Fe(CO) 5 ϩ· or analogous ions provide a measure of the range and magnitude of the internal energy that is deposited into this ion.…”

“…For example, activation of Fe(CO) 5 ϩ· can result in sequential loss of CO molecules with critical energies ranging from 1.15 eV for the loss of the first CO molecule to 7.58 eV for the loss of all five CO molecules; formation of FeC ϩ requires 15.7 eV [43]. The abundances of the fragment ions formed by activation of Fe(CO) 5 ϩ· or analogous ions provide a measure of the range and magnitude of the internal energy that is deposited into this ion. This method, first demonstrated by Kenttämaa and Cooks [42], has been used to characterize the internal energy distribution resulting from ion-surface and ion-gas collisions [43] as well as other activation methods [44,45].…”

A preferred AMPK phosphorylation site adjacent to the inhibitory loop of cardiac and skeletal troponin I

“…The top-down MS strategy allows analysis of intact proteins to obtain a comprehensive assessment and localization of post-translational modifications (PTMs) and point mutations with full sequence coverage and without a priori knowledge. [27][28][29][30][31][32][33][34][35][36][37] It starts with measuring molecular weights of intact proteins followed by fragmentation of the protein in the mass spectrometer with tandem mass spectrometry (MS/MS) such as electron capture dissociation (ECD) 38 to obtain sequence information. ECD has been demonstrated to be especially valuable for mapping phosphorylation sites, because it preserves the labile PTMs during MS/MS process.…”